Synthesis and Biochemical Evaluation of Tritium-Labeled 1-Methyl-N-(8-methyl-8-azabicyclooct-3-yl)-1H-indazole-3-carboxamide, a Useful Radioligand for 5HT3 Receptors

Article abstract of DOI:10.1021/jm00174a013

The advent of potent, highly selective 5HT₃ receptor antagonists has stimulated considerable interest in 5HT₃ receptor mediated physiology and pharmacology.To permit detailed biochemical studies regarding interactions of the indazole class of serotonin (5HT) antagonists with 5HT₃ receptors in multiple tissues, we synthesized 1-methyl-N-(8-methyl-8-azabicyclo<3.2.1>oct-3-yl)-1H-indazole-3-carboxamide (LY278584, compound 9) in high specific activity, tritium-labeled form.This radioligand was selected as a synthetic target because of its potency as a 5HT₃-receptor antagonist, its selectivity for this receptor viz a viz other 5HT-receptor subtypes, and the ability to readily incorporate three tritia via the indazole N-CH3 substituent.Alkylation of N-(8-methyl-8-azabicyclo<3.2.1>oct-3-yl)-1H-indazole-3-carboxamide (8) with sodium hydride and tritium-labeled iodomethane, followed by HPLC purification, resulted in <3H>-9 with a radiochemical purity of 99percent and a specific activity of 80.5 Ci/mmol.This radioligand bound with high affinity to a single class of saturable recognition sites in membranes isolated from cerebral cortex of rat brain.The K_d was 0.69 nM and the B_max was 16.9 fmol/mg of protein.The specific binding was excellent, and accounted for 83-93percent of total binding at concentrations of 2 nM or less.The potencies of known 5HT₃-receptor antagonists as inhibitors of <3H>-9 binding correlated well with their pharmacological receptor affinities as antagonists of 5HT-induced decreases in heart rate and contraction of guinea pig ileum, suggesting the central recognition site for this radioligand may be extremely similar to or identical with pripheral 5HT₃ receptors.